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  • C12P7/06—Ethanol, i.e. non-beverage
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  • C12P7/06—Ethanol, i.e. non-beverage
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
  • C12P7/14—Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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  • C12P7/00—Preparation of oxygen-containing organic compounds
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  • C12P13/00—Preparation of nitrogen-containing organic compounds
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  • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/44—Polycarboxylic acids
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  • C12P7/00—Preparation of oxygen-containing organic compounds
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  • C12P7/54—Acetic acid
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  • C12P7/00—Preparation of oxygen-containing organic compounds
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/58—Aldonic, ketoaldonic or saccharic acids
  • C12P7/60—2-Ketogulonic acid
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the present invention relates to a method for producing a chemical product by continuous fermentation using a fermentation feedstock containing hexose and pentose.
• biodegradable polymer materials such as lactic acid and biofuels such as ethanol have attracted stronger attention as products with sustainability and life cycle assessment (LCA) capability.
• These biodegradable polymer materials and biofuels are generally produced as fermentation products from microorganisms using as a fermentation feedstock glucose, which is a hexose, purified from edible biomass such as maize.
• glucose which is a hexose
• use of edible biomass may cause a rise in its price because of competition with food, resulting in unstable supply of the feedstock.
• attempts are being made to use sugars derived from non-edible biomass such as rice straw as a fermentation feedstock for microorganisms (see Patent Document 1).
• EP 2371 973 A relates to a method for producing a sugar liquid wherein a sugar liquid containing only very small amounts of fermentation-inhibiting substances is produced using a cellulose-containing biomass as a raw material, the method comprising: (1) a step of hydrolyzing a cellulose-containing biomass to produce an aqueous sugar solution; and (2) a step of filtering the obtained aqueous sugar solution through a nanofiltration membrane and/or reverse osmosis membrane to collect a purified sugar liquid from the feed side, while removing fermentation-inhibiting substances from the permeate side.
• EP 1988170 A relates to a method of producing a chemical product through continuous fermentation which includes filtering a culture of a microorganism or cultured cells with a separation membrane to recover a product from a filtrate and simultaneously retaining a nonfiltered fluid in, or refluxing it to, the culture, and adding fermentation materials to the culture, wherein a porous membrane having an average pore size of 0.01 ⁇ m or more to less than 1 ⁇ m is used as the separation membrane and the filtration is conducted with a transmembrane pressure difference in the range of 0.1 to 20 kPa. According to this method, the fermentation productivity of the chemical product can be largely elevated at high stability and a low cost.
• microorganisms that undergo catabolite repression are known as microorganisms capable of fermentation production of biodegradable polymer materials and biofuels.
• continuous fermentation using a mixed sugar of hexose and pentose as a fermentation feedstock for a microorganism results in a remarkably decreased fermentation yield due to catabolite repression.
• the present invention aims to improve the fermentation yield in continuous fermentation using a mixed sugar of hexose and pentose as a fermentation feedstock for a microorganism that undergoes catabolite repression.
• the present inventors discovered that the problem can be solved by a method for producing a chemical product by continuous fermentation using a mixed sugar of hexose and pentose as a fermentation feedstock for a microorganism, wherein a microorganism that undergoes catabolite repression is subjected to continuous fermentation using a separation membrane, thereby reaching the present invention.
• the present invention is as described in (1) to (3) below.
• a chemical product can be produced with a high yield in spite of the fact that a mixed sugar of hexose and pentose is used as a fermentation feedstock for a microorganism(s) that undergo(es) catabolite repression.
• the present invention is a method for fermentative production of a chemical product by culturing a microorganism(s) using a fermentation feedstock, according to Claim 1, which method comprises filtering a culture liquid through a separation membrane; retaining unfiltered liquid in, or refluxing unfiltered liquid to, the culture liquid; adding a fermentation feedstock to the culture liquid; and recovering a product in the filtrate, thereby performing continuous fermentation, wherein the microorganism(s) used is/are a microorganism(s) that undergo(es) catabolite repression, and wherein the fermentation feedstock comprises hexose and pentose.
• the carbon source in the fermentation feedstock comprises a mixed sugar containing pentose and hexose.
• Five-carbon sugar, also called pentose has 5 carbons constituting the sugar.
• Pentose can be classified into aldopentose, which has an aldehyde group at the 1-position, and ketopentose, which has a ketone group at the 2-position. Examples of aldopentose include xylose, arabinose, ribose and lyxose, and examples of ketopentose include ribulose and xylulose.
• the pentose used in the present invention may be any pentose as long as it can be metabolized by a microorganism, and, in view of the abundance in nature, availability and the like, xylose and arabinose are preferred, and xylose is more preferred.
• Hexose has 6 carbons constituting the sugar.
• Hexose can be classified into aldose, which has an aldehyde group at the 1-position, and ketose, which has a ketone group at the 2-position.
• aldose include glucose, mannose, galactose, allose, gulose and talose
• ketose include fructose, psicose and sorbose.
• the hexose used in the present invention may be any hexose as long as it can be metabolized by a microorganism, and, in view of the abundance in nature, availability and the like, glucose, mannose and galactose are preferred, and glucose is more preferred.
• the mixed sugar used in the present invention is not limited, and the mixed sugar is preferably a sugar liquid derived from a cellulose-containing biomass that is known to contain both hexose and pentose.
• the cellulose-containing biomass include herbaceous biomasses such as bagasse, switchgrass, corn stover, rice straw and wheat straw; and woody biomasses such as trees and waste building materials.
• Cellulose-containing biomasses contain cellulose or hemicellulose, which are polysaccharides produced by dehydration condensation of sugars. By hydrolyzing such polysaccharides, sugar liquids which may be used as fermentation feedstocks are produced.
• the method for preparing the sugar liquid derived from a cellulose-containing biomass may be any method, and examples of disclosed methods for producing such a sugar include a method in which a sugar liquid is produced by acid hydrolysis of a biomass using concentrated sulfuric acid ( JP H11-506934 A , JP 2005-229821 A ), and a method in which a biomass is subjected to hydrolysis treatment with dilute sulfuric acid and then enzymatically treated with cellulase and/or the like to produce a sugar liquid ( A.
• examples of disclosed methods in which no acids are used include a method in which a biomass is hydrolyzed using subcritical water at about 250 to 500°C to produce a sugar liquid ( JP 2003-212888 A ), a method in which a biomass is subjected to subcritical water treatment and then enzymatically treated to produce a sugar liquid ( JP 2001-95597 A ), and a method in which a biomass is subjected to hydrolysis treatment with pressurized hot water at 240 to 280°C and then enzymatically treated to produce a sugar liquid ( JP 3041380 B ). These treatments may be followed by purification of the obtained sugar liquid.
• An example of the method is disclosed in WO2010/067785 .
• the weight ratio between the pentose and the hexose contained in the mixed sugar is 1:9 to 9:1 as represented by the ratio of (pentose):(hexose) in terms of the weight ratio between pentose and hexose in the mixed sugar.
• This is the sugar ratio for cases where the mixed sugar is assumed to be a sugar liquid derived from a cellulose-containing biomass.
• the total sugar concentration in the fermentation feedstock used in the present invention is 20 to 300 g/L, and preferably as high as possible within the range in which production of the chemical product by the microorganism(s) is not inhibited. In cases where the total concentration is not more than 20 g/L, the effect of improving the yield from pentose may decrease. Further, in cases where the total sugar concentration is low, the production efficiency of the chemical product also decreases.
• the hexose concentration in the fermentation feedstock used in the present invention is not limited as long as the total sugar concentration and the ratio between pentose and hexose are within the ranges described above.
• a good yield can be obtained even with a mixed sugar liquid containing hexose at a concentration of not less than 5 g/L.
• the fermentation feedstock used in the present invention may preferably be a usual liquid medium containing a carbon source, nitrogen source, inorganic salt, and if necessary, an organic micronutrient(s) such as an amino acid(s) and vitamin(s).
• Examples of the nitrogen source used in the present invention include ammonia gas, aqueous ammonia, ammonium salts, urea and nitric acid salts, and other organic nitrogen sources used supplementarily such as oilcakes, soybean-hydrolyzed liquids, casein digests, other amino acids, vitamins, corn steep liquors, yeasts or yeast extracts, meat extracts, peptides such as peptones, and cells of various fermentation microorganisms and hydrolysates thereof.
• Examples of inorganic salts that may be added as appropriate include phosphoric acid salts, magnesium salts, calcium salts, iron salts and manganese salts.
• the nutrient is added as a preparation or as a natural product containing the nutrient.
• An anti-forming agent is added as required.
• the culture liquid means a liquid obtained as a result of growth of a microorganism(s) in a fermentation feedstock.
• the composition of the fermentation feedstock to be added may be changed as appropriate from the composition of the fermentation feedstock used at the beginning of the culture, such that the productivity of the chemical product of interest increases.
• porous membrane used as a separation membrane in the present invention is explained below.
• the porous membrane used in the present invention is not limited as long as it has a function to separate a culture liquid obtained by culturing a microorganism(s) in a stirred culture vessel or a stirred bioreactor from the microorganism(s) by filtration.
• porous membranes that may be used include porous ceramic membranes, porous glass membranes, porous organic polymer membranes, metal fiber textiles, and non-woven fabrics. Among these, porous organic polymer membranes and ceramic membranes are especially preferred.
• the constitution of the porous membrane used as the separation membrane in the present invention is explained below.
• the porous membrane used in the present invention has a separation performance and a permeability suitable for the properties and use of the liquid to be processed.
• the porous membrane is preferably a porous membrane comprising a porous resin layer in view of the blocking performance, permeability and separation performance, for example, resistance to dirt.
• the porous membrane comprising a porous resin layer preferably has the porous resin layer that functions as a separation functional layer on the surface of a porous base material.
• the porous base material supports the porous resin layer to give strength to the separation membrane.
• the porous base material may be impregnated with the porous resin layer or may not be impregnated with the porous resin layer, which may be selected depending on the use of the membrane.
• the average thickness of the porous base material is preferably 50 ⁇ m to 3000 ⁇ m.
• the porous base material is composed of an organic material and/or inorganic material etc., and an organic fiber is preferably used.
• Preferred examples of the porous base material include woven fabrics and non-woven fabrics composed of organic fibers such as cellulose fibers, cellulose triacetate fibers, polyester fibers, polypropylene fibers and polyethylene fibers. More preferably, a non-woven fabric is used since its density can be relatively easily controlled; it can be simply produced; and it is inexpensive.
• an organic polymer membrane may be preferably used as the porous resin layer.
• the material of the organic polymer membrane include polyethylene resins, polypropylene resins, polyvinyl chloride resins, polyvinylidene fluoride resins, polysulfone resins, polyethersulfone resins, polyacrylonitrile resins, cellulose resins and cellulose triacetate resins.
• the organic polymer membrane may be a mixture of resins containing one or more of these resins as the major component.
• the major component herein means that the component is contained in an amount of not less than 50% by weight, preferably not less than 60% by weight.
• Preferred examples of the material of the organic polymer membrane include those which can be easily formed by solutions and are excellent in physical durability and chemical resistance, such as polyvinyl chloride resins, polyvinylidene fluoride resins, polysulfone resins, polyethersulfone resins and polyacrylonitrile resins.
• a polyvinylidene fluoride resin or a resin containing it as the major component is most preferably used.
• polyvinylidene fluoride resin a homopolymer of vinylidene fluoride is preferably used. Further, as the polyvinylidene fluoride resin, a copolymer with vinyl monomers capable of copolymerizing with vinylidene fluoride is also preferably used. Examples of the vinyl monomers capable of copolymerizing with vinylidene fluoride include tetrafluoroethylene, hexafluoropropylene and ethylene fluoride trichloride.
• the porous membrane that may be used as the separation membrane in the present invention is not limited as long as the microorganism(s) used for fermentation cannot pass through the membrane, and the membrane is preferably selected within the range in which secretions from the microorganism(s) used in the fermentation or particles in the fermentation feedstock do not cause clogging and the filtration performance is stably maintained for a long period. Therefore, the average pore size of the porous separation membrane is preferably not less than 0.01 ⁇ m and less than 5 ⁇ m.
• the average pore size is more preferably not less than 0.01 ⁇ m and less than 1 ⁇ m since, within this range, both a high blocking performance which does not allow leakage of microorganisms and a high permeability can be achieved, and the permeability can be maintained with higher accuracy and reproducibility for a long time.
• the average pore size of the porous membrane is preferably less than 1 ⁇ m.
• the average pore size of the porous membrane is preferably not too large as compared to the size of the microorganism(s).
• the average pore size is preferably not more than 0.4 ⁇ m, more preferably less than 0.2 ⁇ m.
• the microorganism(s) may produce substances other than the chemical product of interest, e.g. substances that are likely to aggregate such as proteins and polysaccharides. Further, in some cases, death of a part of the microorganism(s) in the fermentation culture liquid may produce cell debris. In order to prevent clogging of the porous membrane due to these substances, the average pore size is still more preferably not more than 0.1 ⁇ m.
• the average pore size of the porous membrane in the present invention is preferably not less than 0.01 ⁇ m, more preferably not less than 0.02 ⁇ m, still more preferably not less than 0.04 ⁇ m.
• the average pore size can be determined by measuring the diameters of all pores which can be observed within an area of 9.2 ⁇ m x 10.4 ⁇ m under a scanning electron microscope at a magnification of 10,000x, and then averaging the measured values.
• the average pore size can be determined by taking a picture of the membrane surface under a scanning electron microscope at a magnification of 10,000x, and randomly selecting not less than 10 pores, preferably not less than 20 pores, followed by measuring the diameters of these pores and calculating the number average.
• a pore In cases where a pore is not circular, its size can be determined by a method in which a circle whose area is equal to the area of the pore (equivalent circle) is determined using an image processing device or the like and then the diameter of the equivalent circle is regarded as the diameter of the pore.
• the standard deviation ⁇ of the average pore size of the porous membrane used in the present invention is preferably not more than 0.1 ⁇ m.
• the standard deviation ⁇ of the average pore size is preferably as small as possible.
• the porous membrane used in the present invention permeability to the fermentation culture liquid is one of the important performances.
• the pure water permeability coefficient of the porous membrane before use can be employed.
• the pure water permeability coefficient of the porous membrane is preferably not less than 5.6 x 10 -10 m 3 /m 2 /s/pa when calculated by measuring the amount of permeation of water with a head height of 1 m using purified water at a temperature of 25°C prepared with a reverse osmosis membrane.
• the surface roughness is the average of the height in the direction vertical to the surface.
• the membrane surface roughness is a factor that influences how easily a microorganism attached to the surface of a separation membrane is detached by the effect of washing the membrane surface with flowing liquid generated by stirring or a circulating pump.
• the surface roughness of the porous membrane is not limited as long as it is within the range in which the microorganism(s) and other solids attached to the membrane can be detached.
• the surface roughness is preferably not more than 0.1 ⁇ m. In cases where the surface roughness is not more than 0.1 ⁇ m, the microorganism(s) and other solids attached to the membrane can be easily detached.
• a porous membrane having a membrane surface roughness of not more than 0.1 ⁇ m, an average pore size of not less than 0.01 ⁇ m and less than 1 ⁇ m, and a pure water permeability coefficient of not less than 2 x 10 -9 m 3 /m 2 /s/pa.
• the surface roughness of the porous membrane is not more than 0.1 ⁇ m, the shear force generated on the membrane surface during filtration of the microorganism(s) can be reduced, and hence destruction of the microorganism(s) can be suppressed, and clogging of the porous membrane can also be suppressed.
• the surface roughness of the porous membrane is not more than 0.1 ⁇ m, continuous fermentation can be carried out with a smaller transmembrane pressure difference. Therefore, even in cases where clogging of the porous membrane has occurred, a better washing recovery performance can be obtained as compared to cases where the operation was carried out with a larger transmembrane pressure difference. Since suppression of clogging of the porous membrane allows stable continuous fermentation, the surface roughness of the porous membrane is preferably as small as possible.
• the membrane surface roughness of the porous membrane herein is measured using the following atomic force microscope (AFM) under the following conditions.
• the membrane sample was soaked in ethanol at room temperature for 15 minutes and then soaked in RO water for 24 hours to wash it, followed by drying in the air.
• the RO water means water prepared by filtration through a reverse osmosis membrane (RO membrane), which is a type of filtration membrane, to remove impurities such as ions and salts.
• RO membrane reverse osmosis membrane
• the pore size of the RO membrane is not more than about 2 nm.
• the shape of the porous membrane used in the present invention is preferably a flat membrane.
• its average thickness is selected depending on its use.
• the average thickness in the cases where the shape of the porous membrane is a flat membrane is preferably 20 ⁇ m to 5000 ⁇ m, more preferably 50 ⁇ m to 2000 ⁇ m.
• the shape of the porous membrane used in the present invention is preferably a hollow fiber membrane.
• the inner diameter of the hollow fiber is preferably 200 ⁇ m to 5000 ⁇ m, and the membrane thickness is preferably 20 ⁇ m to 2000 ⁇ m.
• a fabric or knit produced by forming organic fibers or inorganic fibers into a cylindrical shape may be contained in the hollow fiber.
• the porous membrane described above can be produced by, for example, the production method described in WO2007/097260 .
• the separation membrane in the present invention may be a membrane containing at least a ceramic.
• the ceramic in the present invention means a substance that contains a metal oxide and was baked by heat treatment at high temperature.
• the metal oxide include alumina, magnesia, titania and zirconia.
• the separation membrane may be formed by only a metal oxide(s), or may contain silica and/or silicon carbide, and/or mullite and/or cordierite, which are compounds of silica and a metal oxide(s).
• Components forming the separation membrane other than the ceramic are not limited as long as the components can form a porous body as a separation membrane.
• the shape of the separation membrane is not limited, and may be any of a monolith membrane, flat membrane, tubular membrane and the like.
• the separation membrane preferably has a columnar shape in which a penetrating hole(s) is/are formed in the longitudinal direction.
• the separation membrane is preferably a monolith membrane.
• the separation membrane preferably has a penetrating hole(s) in the longitudinal direction.
• modularization of the separation membrane is possible by selecting a preferred mode from the external-pressure type and the internal-pressure type, and filtration can be carried out with the module.
• the side in which the separation membrane contacts with the fermentation culture liquid is hereinafter referred to as the primary side
• the side in which a filtrate containing a chemical product is obtained by filtration is hereinafter referred to as the secondary side.
• the inner-pressure type separation membrane needs to have an inlet and an outlet for the fermentation culture liquid.
• the inlet and the outlet are preferably in a state where they are arranged on a straight line to form a penetrating hole since the flow resistance is small in such a case.
• the container containing the separation membrane can be made thin. A thin separation membrane module is preferred in view of production and handling.
• the porosity of the separation membrane is not limited, but in cases where the porosity is too low, the filtration efficiency is low; and in cases where the porosity is too high, the strength is low. In order to achieve both high filtration efficiency and high strength of the separation membrane, as well as resistance to repeated steam sterilization, the porosity is preferably 20% to 60%.
• the average pore size of the separation membrane is preferably 0.01 ⁇ m to 1 ⁇ m, and a membrane having an average pore size within this range is less likely to be clogged and has excellent filtration efficiency. Further, with an average pore size within the range of 0.02 ⁇ m to 0.2 ⁇ m, substances that easily cause clogging of a separation membrane, such as by-products of fermentation by the microorganism or cultured cells, including proteins and polysaccharides, and cell debris produced by death of the microorganism/cultured cells in the culture liquid, become less likely to cause clogging, which is especially preferred.
• a modular container be provided for collecting the filtrate and that the separation membrane be packed into the container to form a module to be used.
• One or more separation membranes are packed into one module.
• the modular container is preferably composed of a material resistant to repeated steam sterilization.
• the material resistant to steam sterilization include stainless steels, and ceramics having low average porosities.
• Such a ceramic membrane module can be produced by, for example, the production method described in WO2012/086763 , or a commercially available module may be used. Specific examples of the commercially available module include MEMBRALOX Microfiltration Membrane (Pall Corporation) and a ceramic membrane filter Cefilt MF Membrane (NGK Insulators, Ltd.).
• the transmembrane pressure difference during filtration is not limited as long as the fermentation culture liquid can be filtered.
• the structure of the organic polymer membrane is highly likely to be destroyed, and therefore the capacity to produce a chemical product may be deteriorated.
• the transmembrane pressure difference is less than 0.1 kPa, a sufficient amount of permeate of the fermentation culture liquid may not be obtained, and the productivity in production of the chemical product tends to be low.
• the transmembrane pressure difference which is the filtration pressure
• the transmembrane pressure difference is preferably within the range of 0.1 kPa to 150 kPa since, in such a case, the amount of permeate of the fermentation culture liquid can be large, and the decrease in the capacity to produce a chemical product due to destruction of the membrane structure does not occur. Therefore, the capacity to produce a chemical product can be kept high in such a case.
• the transmembrane pressure difference is more preferably within the range of 0.1 kPa to 50 kPa, still more preferably within the range of 0.1 kPa to 20 kPa.
• the transmembrane pressure difference during filtration is not limited as long as the fermentation culture liquid can be filtered.
• the transmembrane pressure difference is preferably not more than 500 kPa. In cases where the operation is carried out at not less than 500 kPa, clogging of the membrane may occur to cause a trouble in the operation of continuous fermentation.
• a siphon using the liquid level difference (hydraulic head difference) between the fermentation culture liquid and the liquid processed through the porous membrane, or a cross-flow circulating pump may be used to generate the transmembrane pressure difference in the separation membrane.
• a suction pump may be placed in the secondary side of the separation membrane.
• the transmembrane pressure difference can be controlled by the suction pressure.
• the transmembrane pressure difference can also be controlled by the pressure of the gas or liquid which is used for introducing the pressure into the fermentation liquid side.
• the difference between the pressure in the fermentation liquid side and the pressure in the side of the liquid processed through the porous membrane can be regarded as the transmembrane pressure difference, and can be used for controlling the transmembrane pressure difference.
• the concentration of pentose in the total amount of filtrate that has passed through the separation membrane is kept at not more than 5 g/l.
• the culture medium is continuously utilized for the fermentation. Therefore, the microorganism(s) undergo(es) catabolite repression more continuously than in cases of batch fermentation. As a result, only pentose remains in a large amount in the culture liquid, and the production yield decreases.
• the pentose concentration in the total amount of filtrate can be kept at not more than 5 g/l even in cases where a microorganism(s) that undergo(es) catabolite repression is/are used.
• the production yield of the chemical product can be increased compared to continuous fermentation without using a separation membrane.
• pentose remains at not less than 5 g/l in the total amount of filtrate obtained by using a separation membrane, the effect of increasing the yield from pentose may decrease, resulting in a decreased production yield.
• the production yield herein means the production yield in continuous fermentation, and is calculated according to the (Equation 3) below, wherein the amount of the chemical product (g) produced by consumption of a carbon source material during a certain period is divided by the amount of the carbon source fed (g) during the period. In this calculation, the sugar that was not utilized for production of the product is also included in the amount of the carbon source fed.
• Production yield g / g Amount of product g / Amount of carbon source fed g
• the concentration of pentose in the total amount of filtrate can be controlled by culture conditions. For example, by changing the sugar concentration in the fermentation feedstock, the sugar supply rate and/or the dilution rate, the concentration of pentose in the total amount of filtrate can be reduced. Alternatively, by increasing a nutrient(s) contained in the fermentation feedstock, consumption of sugar by the microorganism(s) can be increased, and the concentration of pentose in the total amount of filtrate can be reduced.
• the pH and the temperature during fermentation culture of the microorganism(s) are not limited as long as they are within the ranges in which the microorganism(s) can grow.
• the culture is preferably carried out at a pH within the range of 4 to 8 and a temperature within the range of 20 to 75°C.
• the pH of the culture liquid is adjusted in advance with an inorganic or organic acid, alkaline substance, urea, calcium carbonate, ammonia gas or the like to a predetermined pH within the range of, usually, 4 to 8.
• the feed rate of oxygen may be increased by, for example, maintaining the oxygen concentration at not less than 21% by adding oxygen into the air, pressurizing the culture liquid, increasing the stirring rate, and/or increasing the aeration rate.
• continuous fermentation filtration of culture liquid
• microorganism cells may be seeded at high concentration, and continuous fermentation may then be carried out from the beginning of the culture.
• supply of the culture medium and filtration of the culture liquid may be carried out from an appropriate timing(s). The timings of beginning of the supply of the culture medium and filtration of the culture liquid do not necessarily need to be the same.
• the supply of the culture medium and filtration of the culture liquid may be carried out either continuously or intermittently.
• a nutrient(s) necessary for growth of the microorganism cells may be added to the raw culture liquid to allow continuous growth of the cells.
• the microorganism concentration in the culture liquid is preferably maintained such that the productivity of the chemical product is kept high.
• a good production efficiency can be obtained by maintaining the microorganism concentration in the culture liquid at, for example, not less than 5 g/L in terms of the dry weight.
• the microorganism concentration in the culture vessel may be controlled by removing a part of the culture liquid containing the microorganism(s) from the fermenter and then diluting the culture liquid in the vessel with a culture medium.
• the microorganism concentration in the fermenter is too high, clogging of the separation membrane is likely to occur. The clogging may be avoided by removing a part of the culture liquid containing the microorganism(s) and then diluting the culture liquid in the fermenter with the culture medium.
• the performance for producing the chemical product may change depending on the microorganism concentration in the fermenter.
• the production performance may be maintained by removing a part of the culture liquid containing the microorganism(s) and then diluting the culture liquid in the fermenter with a culture medium, using the production performance as an index.
• the number of fermenters is not limited as long as the continuous fermentation culture is carried out by growing microorganism cells while allowing the cells to produce the product.
• the fermentation production rate in batch culture can be determined by dividing the amount of the product (g) upon complete consumption of the carbon source in the feedstock by the time (hr) required for the consumption of the carbon source and the volume (L) of the fermentation culture liquid at that time.
• the yield in the continuous culture can be calculated according to Equation (5) below by dividing the amount of chemical product (g) produced by consumption of the carbon source in the feedstock during a predetermined period by the value obtained by subtracting the amount of carbon source unused by the microorganism(s) (g) from the amount of carbon source fed (g) during this period.
• the yield as used in the present description means this unless otherwise specified.
• the continuous fermentation apparatus used in the present invention is not limited as long as it is an apparatus for producing a chemical product by continuous fermentation in which a fermentation culture liquid of a microorganism(s) is filtered through a separation membrane and the product is recovered from the filtrate, while the unfiltered liquid is retained in, or refluxed to, the fermentation culture liquid; a fermentation feedstock is added to the fermentation culture liquid; and the product in the filtrate is recovered.
• a fermentation culture liquid of a microorganism(s) is filtered through a separation membrane and the product is recovered from the filtrate, while the unfiltered liquid is retained in, or refluxed to, the fermentation culture liquid; a fermentation feedstock is added to the fermentation culture liquid; and the product in the filtrate is recovered.
• Specific examples of the apparatus in which an organic polymer membrane is used include the apparatus described in WO2007/097260 .
• Specific examples of the apparatus in which a ceramic membrane is used include the apparatus described in WO2012/086763 .
• microorganism(s) that may be used in the method for producing a chemical product of the present invention is explained below.
• the microorganism that undergoes catabolite repression used in the present invention generally means a microorganism which metabolizes pentose and whose consumption of pentose is suppressed when fermentation is carried out with a fermentation feedstock comprising a mixed sugar containing hexose and pentose. More specifically, in the present invention, "a microorganism that undergoes catabolite repression" refers to a microorganism which is capable of metabolizing glucose and xylose and whose consumption of xylose is slower in a culture medium comprising a mixed sugar containing glucose and xylose than in a culture medium containing xylose alone when it is cultured by batch culture.
• Xylose consumption rate g / L / hr Total amount of xylose g contained in the fermentation feedstock at the beginning of culture / Time hr required for complete consumption of the xylose contained in the fermentation feedstock after the beginning of culture / Amount of fermentation liquid L
• the rate of consumption of xylose in a culture medium comprising a mixed sugar containing glucose and xylose means the rate of consumption of xylose in the presence of glucose in a culture medium comprising a mixed sugar containing glucose and xylose, and is calculated according to the (Equation 7) below.
• Xylose consumption rate g / L / hr Amount of xylose g consumed from the beginning of culture to the time T / Length of time hr from the beginning of culture to the time T / Amount of fermentation liquid L
• the weight ratio between glucose and xylose in the mixed sugar is set as 1:1.
• the sugar concentration is not limited as long as the sugar can be completely consumed by the microorganism without leaving residual sugar.
• the sugar concentration in the culture medium containing xylose alone is set as the same as the total sugar concentration in the culture medium containing the mixed sugar containing glucose and xylose.
• the time T is the time when glucose has been completely consumed.
• the time T can be determined by measuring the glucose concentration in sampled culture liquid by HPLC, or using a kit or sensor. In cases where complete consumption of glucose occurred later than complete consumption of xylose, the time T is defined as the time when xylose was completely consumed. Also in such cases, the time T can be determined by measuring the xylose concentration in the same manner as in the method of measuring the glucose concentration. In cases where the amount of fermentation liquid has changed by addition of a neutralizer or by sampling during the culture, the calculation is carried out in consideration of the amount of liquid added to the culture liquid or the amount of liquid decreased.
• the microorganism that undergoes catabolite repression is selected from yeasts such as baker's yeast; bacteria such as E. coli and corynebacteria; filamentous fungi; actinomycetes; and the like; which are often used in the fermentation industry.
• microorganisms that may be selected include yeasts such as Pichia, Candida, Pachysolen, Kluyveromyces, Hansenula, Torulopsis, Debaryomyces, Issachenkia, Brettanomyces, Lindnera and Wickerhamomyces; enterobacteria such as Clostridium, Enterobacter, Escherichia and Klebsiella; lactic acid bacteria such as Lactobacillus and Lactococcus ; actinomycetes such as Actinoplanes, Arthrobacter and Streptomyces; and microorganisms belonging to Bacillus, Paenibacillus, Aerobacter, Ampullariella, Staphylococcus, Thermoanaerobacter or Thermus.
• yeasts such as Pichia, Candida, Pachysolen, Kluyveromyces, Hansenula, Torulopsis, Debaryomyces, Issachenkia, Brettanomyces, Lindnera and Wicker
• the microorganism(s) that undergo(es) catabolite repression may be selected either from microorganisms isolated from the natural environment or from microorganisms that do not originally metabolize pentose but are modified by mutation or genetic recombination such that they metabolize pentose.
• Specific examples of the microorganisms that are modified by genetic recombination such that they metabolize pentose include microorganisms in which a metabolic gene for pentose is introduced or enhanced by genetic recombination.
• metabolic genes for xylose among pentoses include the genes for enzymes such as xylose isomerase, xylose reductase, xylitol dehydrogenase and xylulose kinase, and examples of microorganisms given a xylose metabolic capacity by such genetic recombination include microorganisms described in JP 2006-525029 A , JP 2009-112289 A and JP 2010-504756 A .
• the chemical product produced by the present invention is an amino acid or an alcohol such as ethanol, 1,3-propanediol, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, glycerol, butanol, isobutanol, 2-butanol and isopropanol or an organic acid such as acetic acid, lactic acid, adipic acid, pyruvic acid, succinic acid, malic acid, itaconic acid and citric acid.
• the present invention may also be applied to production of substances such as nucleic acids, such as nucleosides including inosine and guanosine, and nucleotides including inosinic acid and guanylic acid; and diamine compounds such as cadaverine; and enzymes, antibiotics and recombinant proteins which is not covered by the scope of the claims.
• nucleic acids such as nucleosides including inosine and guanosine, and nucleotides including inosinic acid and guanylic acid
• diamine compounds such as cadaverine
• enzymes, antibiotics and recombinant proteins which is not covered by the scope of the claims.
• Lactic acid in the fermentation liquid was quantified under the following HPLC conditions by comparison with standard samples.
• the xylose consumption rate in lactic acid fermentation with a lactic acid fermentation microorganism, the Bacillus coagulans NBRC12714 strain was calculated.
• the culture medium the lactic acid fermentation xylose medium having the composition shown in Table 1 or the lactic acid fermentation mixed-sugar medium 1 shown in Table 2 was used. Sampling was carried out as appropriate.
• the concentrations of glucose and xylose in the culture liquid were measured by the method of Reference Example 1, and the concentration of lactic acid as the product was measured by the method of Reference Example 2.
• the Bacillus coagulans NBRC12714 strain was cultured in 50 mL of a preculture medium (10 g/L polypeptone, 2 g/L yeast extract, 1 g/L magnesium sulfate 7H 2 O) supplemented with calcium carbonate in a flask for 24 hours with shaking (preculture).
• the preculture liquid was inoculated to 1 L of the lactic acid fermentation xylose medium or the lactic acid fermentation mixed-sugar medium 1 purged with nitrogen gas, and batch fermentation was performed under the following conditions.
• Fermentation reaction vessel capacity 2 (L) Temperature adjustment: 50 (°C) Aeration in the reaction vessel (nitrogen gas): 100 (mL/min) Stirring rate in the reaction vessel: 200 (rpm) pH Adjustment: adjusted to pH 7 with 5 N Ca(OH) 2 Sterilization: the culture vessels and media to be used were all subjected to high-pressure steam sterilization by autoclaving at 121°C for 20 min.
• the xylose consumption rates in the lactic acid fermentation xylose medium and the lactic acid fermentation mixed-sugar medium 1 were calculated according to the (Equation 6) and (Equation 7) described above, respectively.
• the calculation results are shown in Table 7. From these results, the Bacillus coagulans NBRC12714 strain was judged to be a microorganism that undergoes catabolite repression.
• the xylose consumption rate in ethanol fermentation with an ethanol fermentation microorganism, the Candida tropicalis NBRC0199 strain was calculated.
• the culture medium the ethanol fermentation xylose medium having the composition shown in Table 3 or the ethanol fermentation mixed-sugar medium 1 shown in Table 4 was used. Sampling was carried out as appropriate.
• the concentrations of glucose and xylose in the culture liquid, and the concentration of ethanol as the product were measured by the method of Reference Example 1.
• the Candida tropicalis NBRC0199 strain was cultured in 2 mL of YPD medium in a test tube at 30°C overnight with shaking (pre-preculture).
• the obtained culture liquid was inoculated to 50 mL of fresh YPD medium, and culture was performed overnight with shaking in a 500-mL baffled Erlenmeyer flask (preculture).
• the preculture liquid was inoculated to 2 L of the ethanol fermentation xylose medium or the ethanol fermentation mixed-sugar medium, and batch culture was performed under the following conditions.
• Fermentation reaction vessel capacity 2 (L) Temperature adjustment: 30 (°C) Aeration in the reaction vessel: 100 (mL/min) Stirring rate in the reaction vessel: 800 (rpm) pH Adjustment: none Sterilization: the culture vessels and media to be used were all subjected to high-pressure steam sterilization by autoclaving at 121°C for 20 min.
• the xylose consumption rates in the ethanol fermentation xylose medium and the ethanol fermentation mixed-sugar medium 1 were calculated according to the (Equation 6) and (Equation 7) described above, respectively.
• the calculation results are shown in Table 7. From these results, the Candida tropicalis NBRC0199 strain was judged to be a microorganism that undergoes catabolite repression.
• the xylose consumption rate in 2,3-butanediol fermentation with a 2,3-butanediol fermentation microorganism, the Paenibacillus polymyxa ATCC12321 strain was calculated.
• the 2,3-butanediol fermentation xylose medium having the composition shown in Table 5 or the 2,3-butanediol fermentation mixed-sugar medium 1 shown in Table 6 was used as the culture medium. Sampling was carried out as appropriate, and the concentrations of glucose and xylose in the culture liquid, and the concentration of 2,3-butanediol as the product were measured by the method of Reference Example 1.
• the Paenibacillus polymyxa ATCC12321 strain was cultured in 50 mL of a preculture medium (5 g/L glucose, 5 g/L peptone, 3 g/L yeast extract, 3 g/L malt extract) in a test tube with shaking for 24 hours (preculture).
• the preculture liquid was inoculated to 1 L of the 2,3-butanediol fermentation xylose medium or the 2,3-butanediol fermentation mixed-sugar medium, and batch culture was performed under the following conditions.
• Fermentation reaction vessel capacity 2 (L) Temperature adjustment: 30 (°C) Aeration in the reaction vessel: 100 (mL/min) Stirring rate in the reaction vessel: 800 (rpm) pH Adjustment: adjusted to pH 6.5 with 5 N NaOH Sterilization: the culture vessels and media to be used were all subjected to high-pressure steam sterilization by autoclaving at 121°C for 20 min.
• the xylose consumption rates in the 2,3-butanediol fermentation xylose medium and the 2,3-butanediol fermentation mixed-sugar medium 1 were calculated according to (Equation 6) and (Equation 7) described above, respectively.
• the calculation results are shown in Table 7. From these results, the Paenibacillus polymyxa ATCC12321 strain was judged to be a microorganism that undergoes catabolite repression.
• the Bacillus coagulans NBRC12714 strain was used, and, as a culture medium, the lactic acid fermentation medium having the composition shown in Table 8 was used.
• the Bacillus coagulans NBRC12714 strain was cultured in 50 mL of a preculture medium (10 g/L polypeptone, 2 g/L yeast extract, 1 g/L magnesium sulfate 7H 2 O) supplemented with calcium carbonate in a flask for 24 hours with shaking (preculture).
• the preculture liquid was inoculated to 1 L of the lactic acid fermentation medium purged with nitrogen gas, and batch culture was performed for 96 hours under the conditions of Reference Example 3 (Table 11).
• Lactic acid fermentation medium Glucose 100 g Yeast extract 5 g Ammonium sulfate 1 g K 2 HPO 4 0.4 g Unit (1/Liter)
• the Bacillus coagulans NBRC12714 strain was subjected to batch culture for 128 hours using the lactic acid fermentation mixed-sugar medium shown in Table 2 under the same conditions as in Comparative Example 1 (Table 11).
• Fermentation reaction vessel capacity 2 (L) Temperature adjustment: 50 (°C) Aeration in the reaction vessel (nitrogen gas): 100 (mL/min) Stirring rate in the reaction vessel: 200 (rpm) pH Adjustment: adjusted to pH 7 with 5 N Ca(OH) 2 Amount of the fermentation liquid collected: 3 (L/day) Sterilization: the culture vessels and media to be used were all subjected to high-pressure steam sterilization by autoclaving at 121°C for 20 min.
• Example 1 Production of L-Lactic Acid by Continuous Culture of Bacillus coagulans Using Mixed Sugar (Glucose, Xylose) as Fermentation Feedstock, with Use of Separation Membrane 1
• Fermentation reaction vessel capacity 2 (L) Separation membrane used: polyvinylidene fluoride filtration membrane Effective filtration area of the membrane separation element: 473 (cm 2 ) Temperature adjustment: 50 (°C) Aeration in the fermentation reaction vessel (nitrogen gas): 100 (mL/min) Stirring rate in the fermentation reaction vessel: 200 (rpm) Amount of the fermentation liquid collected: 3 (L/day) Sterilization: the culture vessels comprising the separation membrane element, and the culture media to be used were all subjected to high-pressure steam sterilization by autoclaving at 121°C for 20 min.
• the membrane used was a membrane having the following properties, and the transmembrane pressure difference during filtration was allowed to change within the range of 0.1 to 20 kPa.
• Average pore size 0.1 ⁇ m Standard deviation of the average pore size: 0.035 ⁇ m
• Membrane surface roughness 0.06 ⁇ m
• Pure water permeation coefficient 50 x 10 -9 m 3 /m 2 /s/pa
• Example 2 Continuous Culture of Bacillus coagulans Using Mixed Sugar (Glucose, Xylose) as Fermentation Feedstock, with Use of Separation Membrane 2
• the Candida tropicalis NBRC0199 strain was used as an ethanol fermentation microorganism, and the ethanol fermentation medium having the composition shown in Table 12 was used as the culture medium.
• the Candida tropicalis NBRC0199 strain was cultured in 2 mL of YPD medium in a test tube at 30°C overnight with shaking (pre-preculture).
• the obtained culture liquid was inoculated to 50 mL of fresh YPD medium, and culture was performed overnight with shaking in a 500-mL baffled Erlenmeyer flask (preculture).
• the preculture liquid was inoculated to 1.5 L of the ethanol fermentation medium, and batch culture was performed for 16 hours under the following conditions to produce ethanol (Table 14).
• Fermentation reaction vessel capacity 2 (L) Temperature adjustment: 30 (°C) Aeration in the reaction vessel: 100 (mL/min) Stirring rate in the reaction vessel: 800 (rpm) pH Adjustment: none Sterilization: the culture vessels and media to be used were all subjected to high-pressure steam sterilization by autoclaving at 121°C for 20 min.
• Ethanol fermentation medium Glucose 70 g Peptone 20 g Yeast extract 10 g Unit (1/Liter)
• the Candida tropicalis NBRC0199 strain was cultured in 2 mL of YPD medium in a test tube at 30°C overnight with shaking (pre-pre-preculture).
• the obtained culture liquid was inoculated to 50 mL of fresh YPD medium, and culture was performed overnight with shaking in a 500-mL baffled Erlenmeyer flask (pre-preculture).
• the pre-preculture liquid was inoculated to 1.5 L of the ethanol fermentation mixed-sugar medium 2 in a continuous fermentation apparatus, and culture was carried out for 16 hours with stirring at 800 rpm by the stirrer attached to the fermentation reaction vessel while the aeration rate and the temperature in the fermentation reaction vessel were controlled (preculture).
• operation of a pump for collecting the fermentation liquid was started, and the culture medium was continuously supplied. While the amount of collection of culture liquid containing the microorganism was controlled such that the amount of fermentation liquid in the continuous fermentation apparatus was 1.5 L, continuous culture was performed for 295 hours under the following conditions to produce ethanol (Table 14).
• Fermentation reaction vessel capacity 2 (L) Temperature adjustment: 30 (°C) Aeration in the fermentation reaction vessel: 100 (mL/min) Stirring rate in the fermentation reaction vessel: 800 (rpm) pH Adjustment: none Amount of the fermentation liquid collected: 1 (L/day) Sterilization: the culture vessels and media to be used were all subjected to high-pressure steam sterilization by autoclaving at 121°C for 20 min.
• Example 4 Production of Ethanol by Continuous Culture of Candida tropicalis Using Mixed Sugar (Glucose, Xylose) as Fermentation Feedstock, with Use of Separation Membrane
• the separation membrane element employed was in a flat-membrane shape.
• the Candida tropicalis NBRC0199 strain was cultured in 2 mL of YPD medium in a test tube at 30°C overnight (pre-pre-preculture).
• the obtained culture liquid was inoculated to 50 mL of fresh YPD medium, and culture was performed overnight with shaking in a 500-mL baffled Erlenmeyer flask (pre-preculture).
• the pre-preculture liquid was inoculated to 1.5 L of the ethanol fermentation mixed-sugar medium 2 in a continuous fermentation apparatus, and culture was carried out for 36 hours with stirring at 800 rpm by the stirrer attached to the fermentation reaction vessel while the aeration rate and the temperature in the fermentation reaction vessel were controlled (preculture).
• operation of a pump for circulating the fermentation liquid was started, and the culture medium was continuously supplied. While the amount of culture liquid filtered was controlled such that the amount of fermentation liquid in the continuous fermentation apparatus was 1.5 L, continuous culture was performed for 300 hours under the following conditions to produce ethanol (Table 14).
• Fermentation reaction vessel capacity 2 (L) Separation membrane used: polyvinylidene fluoride filtration membrane Effective filtration area of the membrane separation element: 120 (cm 2 ) Temperature adjustment: 30 (°C) Aeration in the fermentation reaction vessel: 100 (mL/min) Stirring rate in the fermentation reaction vessel: 800 (rpm) pH Adjustment: none Amount of the fermentation liquid collected: 1 (L/day) Sterilization: the culture vessels comprising the separation membrane element, and the culture media to be used were all subjected to high-pressure steam sterilization by autoclaving at 121°C for 20 min.
• the membrane used was a membrane having the same properties as in Example 1, and the transmembrane pressure difference during filtration was allowed to change within the range of 0.1 to 19.8 kPa.
• [Table 14] (Comparative Example 4) (Comparative Example 5) (Comparative Example 6) (Example 4) Fermentation period (hr) 16 23 295 300 Total glucose fed (g) 70 30 370 375 Total xylose fed (g) 0 40 490 500 Total production of ethanol (g) 32 17 215 290 Unused glucose (g) 0 0 0 0 Unused xylose (g) 0 0 50 13 Unused xylose / total amount of filtrate (g/L) 0 0 4 1 Yield (g/g) 0.46 0.24 0.27 0.34
• the Paenibacillus polymyxa ATCC12321 strain which is a 2,3-butanediol microorganism, and the 2,3-butanediol fermentation medium having the composition shown in Table 15 as the culture medium, were used.
• the Paenibacillus polymyxa ATCC12321 strain was cultured in 50 mL of a preculture medium (5 g/L glucose, 5 g/L peptone, 3 g/L yeast extract, 3 g/L malt extract) in a test tube with shaking for 24 hours (preculture).
• the preculture liquid was inoculated to 1 L of the 2,3-butanediol fermentation medium, and batch culture was performed under the following conditions for 27 hours to produce 2,3-butanediol (Table 17).
• Fermentation reaction vessel capacity 2 (L) Temperature adjustment: 30 (°C) Aeration in the reaction vessel: 100 (mL/min) Stirring rate in the reaction vessel: 800 (rpm) pH Adjustment: adjusted to pH 6.5 with 5 N NaOH Sterilization: the culture vessels and media to be used were all subjected to high-pressure steam sterilization by autoclaving at 121°C for 20 min.
• Fermentation reaction vessel capacity 2 (L) Temperature adjustment: 30 (°C) Aeration in the reaction vessel: 100 (mL/min) Stirring rate in the reaction vessel: 800 (rpm) pH Adjustment: adjusted to pH 6.5 with 5 N NaOH Amount of the fermentation liquid collected: 0.6 (L/day) Sterilization: the culture vessels and media to be used were all subjected to high-pressure steam sterilization by autoclaving at 121°C for 20 min.
• Fermentation reaction vessel capacity 2 (L) Separation membrane used: polyvinylidene fluoride filtration membrane Effective filtration area of the membrane separation element: 120 (cm 2 ) Temperature adjustment: 30 (°C) Aeration in the fermentation reaction vessel: 100 (mL/min) Stirring rate in the fermentation reaction vessel: 800 (rpm) Amount of the fermentation liquid collected: 0.6 L/day Sterilization: the culture vessels comprising the separation membrane element, and the culture media to be used were all subjected to high-pressure steam sterilization by autoclaving at 121°C for 20 min.
• the membrane used was a membrane having the same properties as in Example 1, and the transmembrane pressure difference during filtration was allowed to change within the range of 0.1 to 20 kPa.
• the xylose consumption rates in the ethanol fermentation xylose medium and the ethanol fermentation mixed-sugar medium 1 were calculated according to (Equation 6) and (Equation 7) described above, respectively.
• the calculation results are shown in Table 22. From these results, the Pichia stipitis NBRC1687 strain was judged to be a microorganism that undergoes catabolite repression.
• the Candida utilis CuLpLDH strain which was prepared by the method disclosed in WO2010/140602 , was used.
• the culture medium the D-lactic acid fermentation xylose medium having the composition shown in Table 18 or the D-lactic acid fermentation mixed-sugar medium 1 shown in Table 19 was used. Batch culture was performed for 40 hours under the same conditions as in Comparative Example 1 except that the pH was adjusted to 6.0 with 1 N calcium hydroxide, and the rate of consumption of xylose in D-lactic acid fermentation was calculated.
• the xylose consumption rates in the D-lactic acid fermentation xylose medium and the D-lactic acid fermentation mixed-sugar medium 1 were calculated according to (Equation 6) and (Equation 7) described above, respectively. The calculation results are shown in Table 22. From these results, the Candida utilis CuLpLDH strain was judged to be a microorganism that undergoes catabolite repression.
• the culture medium the ethanol fermentation xylose medium 2 having the composition shown in Table 20 or the ethanol fermentation mixed-sugar medium 3 shown in Table 21 was used. Sampling was carried out as appropriate, and the concentrations of glucose and xylose in the culture liquid, and the concentration of ethanol as the product were measured by the method of Reference Example 1.
• the Escherichia coli KO11 strain was cultured in 2 mL of a preculture medium (20 g/L glucose, 10 g/L yeast extract, 5 g/L tryptone, 5 g/L NaCl) in a test tube at 30°C overnight (pre-preculture).
• the obtained culture liquid was inoculated to 50 mL of a preculture medium placed in a 500-mL baffled Erlenmeyer flask, and culture was performed overnight (preculture).
• the preculture liquid was inoculated to 1.5 L of the ethanol fermentation xylose medium 2 or the ethanol fermentation mixed-sugar medium 3, and batch fermentation was carried out for 16 hours under the following operating conditions while the temperature and the pH were controlled.
• Culture vessel capacity 2 (L) Temperature adjustment: 30 (°C) Aeration in the fermentation reaction vessel: 100 (mL/min) Stirring rate in the fermentation reaction vessel: 800 (rpm) pH Adjustment: adjusted to pH 6 with 5 N Ca(OH) 2 Sterilization: the fermentation vessel and media to be used were all subjected to high-pressure steam sterilization by autoclaving at 121°C for 20 min.
• the xylose consumption rates in the ethanol fermentation xylose medium 2 and the ethanol fermentation mixed-sugar medium 3 were calculated according to (Equation 6) and (Equation 7) described above, respectively. The calculation results are shown in Table 22. From these results, the Escherichia coli KO11 strain was judged to be a microorganism that undergoes catabolite repression. [Table 22] (Reference Example 6) (Reference Example 7) (Reference Example 8) Xylose consumption rate in the xylose medium (g/L/hr) 2.22 1.59 1.46 Xylose consumption rate in the mixed-sugar medium (g/L/hr) 0.25 0.09 0.16
• the Candida utilis CuLpLDH strain which was prepared by the method disclosed in WO2010/140602 , was used. Batch culture was carried out for 23 hours under the same conditions as in Comparative Example 4 except that the pH was adjusted to 6.0 with 1 N calcium hydroxide and the D-lactic acid fermentation mixed-sugar medium 2 shown in Table 24 was used, to produce D-lactic acid (Table 25).
• D-Lactic acid fermentation mixed-sugar medium 2 Glucose 20 g Xylose 50 g Yeast extract 10 g Bactopeptone 20 g Unit (1/Liter)
• Continuous fermentation was carried out using the Candida utilis CuLpLDH strain as the microorganism, with use of a separation membrane.
• the continuous fermentation was performed for 310 hours under the same conditions as in Example 4 except that the period of preculture was 50 hours; the pH was adjusted to 6.0 with 1 N calcium hydroxide; and the D-lactic acid fermentation mixed-sugar medium 2 shown in Table 24 was used as the culture medium, to produce D-lactic acid.
• the Escherichia coli KO11 strain was cultured in 2 mL of a preculture medium (20 g/L glucose, 10 g/L yeast extract, 5 g/L tryptone, 5 g/L NaCl) in a test tube at 30°C overnight (pre-preculture).
• the obtained culture liquid was inoculated to 50 mL of a preculture medium placed in a 500-mL baffled Erlenmeyer flask, and culture was performed overnight (preculture).
• the preculture liquid was inoculated to 1 L of the ethanol fermentation medium 2 having the composition shown in Table 26, and batch fermentation was carried out for 16 hours under the following operating conditions while the temperature and the pH were controlled, to produce ethanol (Table 28).
• the Escherichia coli KO11 strain was cultured in 2 mL of a preculture medium (20 g/L glucose, 10 g/L yeast extract, 5 g/L tryptone, 5 g/L NaCl) in a test tube at 30°C overnight (pre-pre-preculture).
• the obtained culture liquid was inoculated to 50 mL of a preculture medium in a 500-mL baffled Erlenmeyer flask, and culture was performed overnight (pre-preculture).
• the pre-preculture liquid was inoculated to the ethanol fermentation mixed-sugar medium 4 having the composition shown in Table 27 placed in a continuous culture apparatus (the same apparatus as shown in Fig. 2 of WO2007/097260 except that the separation membrane element was eliminated), and batch fermentation was carried out for 24 hours under the operating conditions shown below while the temperature and the pH were controlled (preculture).
• continuous culture was started to produce ethanol.
• a Perista BioMini Pump Type AC-2120 was used to supply the culture medium directly to the culture vessel and to collect the culture liquid containing the microorganism directly from the culture vessel.
• the Escherichia coli KO11 strain was cultured in 2 mL of a preculture medium (20 g/L glucose, 10 g/L yeast extract, 5 g/L tryptone, 5 g/L NaCl) in a test tube at 30°C overnight (pre-pre-preculture).
• the obtained culture liquid was inoculated to 50 mL of a preculture medium in a 500-mL baffled Erlenmeyer flask, and culture was performed overnight (pre-preculture).
• the pre-preculture liquid was inoculated to the ethanol fermentation mixed-sugar medium 4 having the composition shown in Table 27 placed in a continuous fermentation apparatus equipped with an integrated membrane having the properties shown below (the apparatus shown in Fig. 2 of WO2007/097260 ), and batch fermentation was carried out for 24 hours under the operating conditions shown below while the temperature and the pH were controlled (preculture).
• continuous culture was started to produce ethanol.
• a Perista BioMini Pump Type AC-2120 (ATTO) was used for supplying the ethanol fermentation mixed-sugar medium 4 having the composition shown in Table 27 and filtering the culture liquid.
• the culture medium was directly supplied to the culture vessel, and the culture liquid was filtered through an element having an immobilized separation membrane.
• Fermentation reaction vessel capacity 2 (L) Separation membrane used: polyvinylidene fluoride filtration membrane Effective filtration area of the membrane separation element: 473 cm 2 Pure water permeation coefficient of the separation membrane: 50 x 10 -9 m 3 /m 2 /s/Pa Average pore size of the separation membrane: 0.1 ⁇ m Standard deviation of the average pore size: ⁇ 0.035 ⁇ m Surface roughness of the separation membrane: 0.06 ⁇ m Temperature adjustment: 30 (°C) pH Adjustment: adjusted to pH 6 with 5 N Ca(OH) 2 Rate of collection of the fermentation liquid: 2 L/day Sterilization: the fermentation vessel comprising the separation membrane element, and media to be used were all subjected to high-pressure steam sterilization by autoclaving at 121°C for 20 min.
• a biomass-derived sugar liquid was used as the fermentation feedstock.
• a cellulose saccharification liquid prepared using a nanofiltration membrane by the preparation method described in Example 2 of WO2010/067785 was used.
• the medium was prepared as shown in Table 29 using reagents as appropriate. Batch culture was performed for 70 hours under the same conditions as in Comparative Example 2 except that the different culture medium was used and 4 N KOH was used as the neutralizer, to produce L-lactic acid (Table 30).
• Lactic acid fermentation sugar liquid medium Glucose 60 g Xylose 20 g Yeast extract 5 g Ammonium sulfate 1 g K 2 HPO 4 0.4 g Unit (1/Liter)
• a biomass-derived sugar liquid was used as the fermentation feedstock.
• a lactic acid fermentation sugar liquid medium the culture medium described in Table 29 was used similarly to Comparative Example 19. Continuous culture was performed for 250 hours under the same conditions as in Comparative Example 3 except that the different culture medium was used and 4 N KOH was used as the neutralizer, to produce lactic acid (Table 30).
• a biomass-derived sugar liquid was used as the fermentation feedstock.
• a lactic acid fermentation sugar liquid medium the culture medium described in Table 29 was used similarly to Comparative Example 19. Continuous culture was performed using a separation membrane for 260 hours under the same conditions as in Example 1 except that the different culture medium was used and 4 N KOH was used as the neutralizer, to produce L-lactic acid.
• a biomass-derived sugar liquid was used as the fermentation feedstock.
• a cellulose saccharification liquid prepared using a nanofiltration membrane by the preparation method described in Example 2 of WO2010/067785 was used.
• the medium was prepared as shown in Table 31 using reagents as appropriate. Batch culture was performed for 23 hours under the same conditions as in Comparative Example 17 except that the different culture medium was used, to produce ethanol (Table 32).
• Ethanol fermentation sugar liquid medium Glucose 30 g Xylose 15 g Yeast extract 10g Tryptone 5g NaCl 5 g Unit (1/Liter)
• the Candida tropicalis NBRC0199 strain Using the Candida tropicalis NBRC0199 strain, continuous fermentation was performed with use of a ceramic separation membrane.
• the culture medium shown in Table 33 was used as the fermentation medium.
• the Candida tropicalis NBRC0199 strain was cultured in 2 mL of YPD medium in a test tube at 30°C overnight with shaking (pre-pre-preculture).
• the obtained culture liquid was inoculated to 50 mL of YPD medium in a 500-mL baffled Erlenmeyer flask, and culture was performed overnight with shaking (pre-preculture).
• the pre-preculture liquid was inoculated to 1.5 L of YPDX medium placed in a membrane-separation-type continuous fermentation apparatus (the apparatus shown in Fig.
• the Paenibacillus polymyxa ATCC12321 strain was cultured in 2 mL of a preculture medium (5 g/L glucose, 5 g/L peptone, 3 g/L yeast extract, 3 g/L malt extract) in a test tube at 30°C overnight (pre-pre-preculture).
• the obtained culture liquid was inoculated to 50 mL of a preculture medium in a 500-mL baffled Erlenmeyer flask, and culture was performed overnight (pre-preculture).
• the pre-preculture liquid was inoculated to the 2,3-butanediol fermentation mixed-sugar medium having the composition shown in Table 16 placed in a continuous culture apparatus (the apparatus shown in Fig. 2 of WO2007/097260 ), and batch fermentation was carried out for 30 hours under the operating conditions shown below while the temperature and the pH were controlled (preculture).
• continuous culture was started using the 2,3-butanediol fermentation mixed-sugar medium having the composition shown in Table 16 to produce 2,3-butanediol. While the transmembrane pressure difference during filtration was controlled at not more than 500 kPa, continuous culture was carried out for 300 hours to produce 2,3-butanediol (Table 35).
• Fermenter capacity 2 (L) Separation membrane used: Celfit microfiltration membrane Monolith ⁇ 4-19 (NGK Insulators, Ltd.) Length of the membrane separation element: 500 mm Average pore size of the separation membrane: 0.1 ⁇ m Temperature adjustment: 30 (°C) Aeration in the fermentation reaction vessel: 100 (mL/min) Stirring rate in the fermentation reaction vessel: 800 (rpm) pH Adjustment: adjusted to pH 6.5 with 5 N Ca(OH) 2 [Table 35]
• Fermentation period (hr) 310 Total glucose fed (g) 129 Total xylose fed (g) 258 Total production of 2,3-butanediol (g) 111 Unused glucose (g) 0 Unused xylose (g) 5 Unused xylose / total amount of filtrate (g/L) 0.8 Yield (g/g) 0.29
• the efficiencies of fermentation production of various chemical products using a fermentation feedstock containing pentose and hexose can be largely increased.

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